Publications by authors named "Hans Hombauer"

19 Publications

  • Page 1 of 1

Ligation of newly replicated DNA controls the timing of DNA mismatch repair.

Curr Biol 2021 03 7;31(6):1268-1276.e6. Epub 2021 Jan 7.

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany; Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg 69120, Germany. Electronic address:

Mismatch repair (MMR) safeguards genome stability through recognition and excision of DNA replication errors. How eukaryotic MMR targets the newly replicated strand in vivo has not been established. MMR reactions reconstituted in vitro are directed to the strand containing a preexisting nick or gap, suggesting that strand discontinuities could act as discrimination signals. Another candidate is the proliferating cell nuclear antigen (PCNA) that is loaded at replication forks and is required for the activation of Mlh1-Pms1 endonuclease. Here, we discovered that overexpression of DNA ligase I (Cdc9) in Saccharomyces cerevisiae causes elevated mutation rates and increased chromatin-bound PCNA levels and accumulation of Pms1 foci that are MMR intermediates, suggesting that premature ligation of replication-associated nicks interferes with MMR. We showed that yeast Pms1 expression is mainly restricted to S phase, in agreement with the temporal coupling between MMR and DNA replication. Restricting Pms1 expression to the G2/M phase caused a mutator phenotype that was exacerbated in the absence of the exonuclease Exo1. This mutator phenotype was largely suppressed by increasing the lifetime of replication-associated DNA nicks, either by reducing or delaying Cdc9 ligase activity in vivo. Therefore, Cdc9 dictates a window of time for MMR determined by transient DNA nicks that direct the Mlh1-Pms1 in a strand-specific manner. Because DNA nicks occur on both newly synthesized leading and lagging strands, these results establish a general mechanism for targeting MMR to the newly synthesized DNA, thus preventing the accumulation of mutations that underlie the development of human cancer.
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http://dx.doi.org/10.1016/j.cub.2020.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8281387PMC
March 2021

Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function.

Commun Biol 2020 12 10;3(1):751. Epub 2020 Dec 10.

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.

Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.
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http://dx.doi.org/10.1038/s42003-020-01481-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730388PMC
December 2020

Extensive 5'-surveillance guards against non-canonical NAD-caps of nuclear mRNAs in yeast.

Nat Commun 2020 11 2;11(1):5508. Epub 2020 Nov 2.

Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, 69120, Heidelberg, Germany.

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5'-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3'-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.
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http://dx.doi.org/10.1038/s41467-020-19326-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606564PMC
November 2020

Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio.

Nucleic Acids Res 2020 01;48(1):264-277

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg D-69120, Germany.

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.
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http://dx.doi.org/10.1093/nar/gkz1006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145683PMC
January 2020

A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance.

Nucleic Acids Res 2019 01;47(1):237-252

DNA Repair Mechanisms and Cancer, German Cancer Research Center (DKFZ), Heidelberg D-69120, Germany.

The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR's overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.
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http://dx.doi.org/10.1093/nar/gky1154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326808PMC
January 2019

Alterations in cellular metabolism triggered by or inactivation cause imbalanced dNTP pools and increased mutagenesis.

Proc Natl Acad Sci U S A 2017 05 17;114(22):E4442-E4451. Epub 2017 Apr 17.

German Cancer Research Center, 69120 Heidelberg, Germany;

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (, , and ) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.
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http://dx.doi.org/10.1073/pnas.1618714114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465912PMC
May 2017

Visualization of mismatch repair complexes using fluorescence microscopy.

DNA Repair (Amst) 2016 Feb 12;38:58-67. Epub 2015 Dec 12.

German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany. Electronic address:

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients. During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5' and 3' nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed. Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms.
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http://dx.doi.org/10.1016/j.dnarep.2015.11.014DOI Listing
February 2016

New insights into the mechanism of DNA mismatch repair.

Chromosoma 2015 Dec 11;124(4):443-62. Epub 2015 Apr 11.

German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany.

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR.
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http://dx.doi.org/10.1007/s00412-015-0514-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600670PMC
December 2015

PCNA and Msh2-Msh6 activate an Mlh1-Pms1 endonuclease pathway required for Exo1-independent mismatch repair.

Mol Cell 2014 Jul 26;55(2):291-304. Epub 2014 Jun 26.

Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Moores-UCSD Cancer Center, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA; Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, CA 92093-0669, USA. Electronic address:

Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1)-dependent and -independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyperaccumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.
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http://dx.doi.org/10.1016/j.molcel.2014.04.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113420PMC
July 2014

Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae.

PLoS Genet 2014 May 8;10(5):e1004327. Epub 2014 May 8.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Moores-UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, California, United States of America; Department of Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.
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http://dx.doi.org/10.1371/journal.pgen.1004327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014439PMC
May 2014

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

PLoS Genet 2013 Oct 31;9(10):e1003869. Epub 2013 Oct 31.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.
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http://dx.doi.org/10.1371/journal.pgen.1003869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814310PMC
October 2013

Checkpoint kinases regulate a global network of transcription factors in response to DNA damage.

Cell Rep 2013 Jul 27;4(1):174-88. Epub 2013 Jun 27.

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

DNA damage activates checkpoint kinases that induce several downstream events, including widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs) are not well understood. Here, we integrate kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics to map kinases and downstream TFs to transcriptional regulatory networks. Specifically, we investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1) in the transcriptional response to DNA damage caused by methyl methanesulfonate. The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network involves at least nine TFs, many of which have Rad53-dependent phosphorylation sites, as regulators of checkpoint-kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network that regulates stress response genes independently of the checkpoint kinases.
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http://dx.doi.org/10.1016/j.celrep.2013.05.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855057PMC
July 2013

Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication.

Science 2011 Dec;334(6063):1713-6

Ludwig Institute for Cancer Research, Departments of Medicine and Cellular and Molecular Medicine and Cancer Center, Moores-UCSD Cancer Center, University of California School of Medicine-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA.

In eukaryotes, it is unknown whether mismatch repair (MMR) is temporally coupled to DNA replication and how strand-specific MMR is directed. We fused Saccharomyces cerevisiae MSH6 with cyclins to restrict the availability of the Msh2-Msh6 mismatch recognition complex to either S phase or G2/M phase of the cell cycle. The Msh6-S cyclin fusion was proficient for suppressing mutations at three loci that replicate at mid-S phase, whereas the Msh6-G2/M cyclin fusion was defective. However, the Msh6-G2/M cyclin fusion was functional for MMR at a very late-replicating region of the genome. In contrast, the heteroduplex rejection function of MMR during recombination was partially functional during both S phase and G2/M phase. These results indicate a temporal coupling of MMR, but not heteroduplex rejection, to DNA replication.
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http://dx.doi.org/10.1126/science.1210770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806717PMC
December 2011

Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.

Cell 2011 Nov;147(5):1040-53

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA.

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.
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http://dx.doi.org/10.1016/j.cell.2011.10.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478091PMC
November 2011

SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder.

Cell 2010 Jul 15;142(2):203-17. Epub 2010 Jul 15.

Neurogenetics Laboratory, Institute for Genomic Medicine, Howard Hughes Medical Institute, Department of Neurosciences and Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.
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http://dx.doi.org/10.1016/j.cell.2010.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940322PMC
July 2010

Cdc28/Cdk1 positively and negatively affects genome stability in S. cerevisiae.

J Cell Biol 2009 May 27;185(3):423-37. Epub 2009 Apr 27.

Department of Medicine, Cancer Center, Ludwig Institute for Cancer Research, University of California, San Diego School of Medicine, La Jolla, CA 92093, USA.

We studied the function of the cyclin-dependent kinase Cdc28 (Cdk1) in the DNA damage response and maintenance of genome stability using Saccharomyces cerevisiae. Reduced Cdc28 activity sensitizes cells to chronic DNA damage, but Cdc28 is not required for cell viability upon acute exposure to DNA-damaging agents. Cdc28 is also not required for activation of the DNA damage and replication checkpoints. Chemical-genetic analysis reveals that CDC28 functions in an extensive network of pathways involved in maintenance of genome stability, including homologous recombination, sister chromatid cohesion, the spindle checkpoint, postreplication repair, and telomere maintenance. In addition, Cdc28 and Mre11 appear to cooperate to prevent mitotic catastrophe after DNA replication arrest. We show that reduced Cdc28 activity results in suppression of gross chromosomal rearrangements (GCRs), indicating that Cdc28 is required for formation or recovery of GCRs. Thus, we conclude that Cdc28 functions in a genetic network that supports cell viability during DNA damage while promoting the formation of GCRs.
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http://dx.doi.org/10.1083/jcb.200811083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700387PMC
May 2009

Generation of active protein phosphatase 2A is coupled to holoenzyme assembly.

PLoS Biol 2007 Jun;5(6):e155

Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria.

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Delta strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.
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http://dx.doi.org/10.1371/journal.pbio.0050155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885835PMC
June 2007

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A.

Curr Biol 2003 Dec;13(23):2058-64

Institute of Medical Biochemistry, Division of Molecular Biology, Vienna Biocenter, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.
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http://dx.doi.org/10.1016/j.cub.2003.10.032DOI Listing
December 2003

A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo.

Genes Dev 2003 Sep;17(17):2138-50

Institute of Medical Biochemistry, Division of Molecular Biology, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria.

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man.
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http://dx.doi.org/10.1101/gad.259903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC196455PMC
September 2003
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